Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5719-2

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Inhibition of Candida albicans virulence factors by novel levofloxacin derivatives Beema Shafreen Raja Mohamed & Muthamil Subramanian & Karutha Pandian Shunmugiah

Received: 7 December 2013 / Revised: 6 March 2014 / Accepted: 22 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Candida albicans is an important opportunistic fungal pathogen, responsible for biofilm associated infections in immunocompromised patients. The aim of the present study was to investigate the antibiofilm properties of novel levofloxacin derivatives on C. albicans biofilms. The levofloxacin derivatives at their Biofilm Inhibitory Concentrations (BIC) were able to inhibit the biofilms of C. albicans, the yeast-to-hyphal transition and were also able to disrupt their mature biofilms. Furthermore, Real-time PCR analysis showed that the expression of ergosterol biosynthesis pathway gene (ERG11) and the efflux pump-encoding genes (CDR1 and MDR1) was decreased upon treatment with the levofloxacin derivatives. The total ergosterol content quantified using UV spectrophotomer showed decrease in ergosterol in the presence of levofloxacin derivatives. Overall, levofloxacin derivatives (6a, 6c and 7d) are capable of inhibiting C. albicans virulence factors. Therefore, these compounds with potential therapeutic implications can be used as new strategy to treat biofilm-related candidal infections. Keywords C. albicans . Levofloxacin derivatives . Ergosterol . Yeast–hyphal . Real-time PCR

Introduction Candida spp. are pathogenic yeasts that cause oral, vaginal and systemic infections and among them Candida albicans Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5719-2) contains supplementary material, which is available to authorized users. B. S. Raja Mohamed : M. Subramanian : K. P. Shunmugiah (*) Department of Biotechnology, Alagappa University, Science Campus, Karaikudi 630 004, Tamil Nadu, India e-mail: [email protected]

has been regarded as the most common causative agent of opportunistic fungal infection in humans (Achkar and Fries 2010; Martin 1999). The factors aiding C. albicans infections include antibiotic and immunosuppressive therapies, human immunodeficiency virus (HIV) infection, diabetes and old age (Bizerra et al. 2008). In addition to bacterial biofilms, biofilms formed by C. albicans on medical devices is the major cause of device-related infections (Chandra et al. 2001b; Kojic and Darouiche 2004). C. albicans biofilm forms a complex threedimensional structure which constitutes different cell types such as yeast, pseudo-hyphae, hyphae and exopolysaccharide (Nett and Andes 2006). This complex three-dimensional organization is responsible for a heterogeneous microenvironment (Bonhomme and d’Enfert 2013) and this heterogeneity of microbial biofilms can have an adverse impact on the response to antimicrobial therapy (El-Azizi et al. 2004). The formation of biofilm also protects C. albicans from host defences and conventional antibiotics by sustaining the required spatial stability and by controlling its own microenvironment (Soll 2008). In C. albicans, sterols are essential for membrane function and organization (Henry et al. 2000). Ergosterol (ERG) is an important sterol component of the yeast cell membrane. ERG plays an essential role during several cellular processes such as membrane transport, sporulation and promotes yeast cell mating by pheromone signals (Zhang et al. 2010). The lanosterol 14-α demethylase (ERG11) involved in sterol biosynthesis is the specific target for azole drugs. Extensive and continuous use of these azole drugs (fluconazole, ketoconazole and itraconazole) has lead to the hasty development of drug resistance in C. albicans (White et al. 2002). During the course of chemotherapy, C. albicans acquired multidrug resistance to those drugs by introducing mutations in ERG11 (Henry et al. 2000; Kohli et al. 2002), which in turn reduced the efficiency of the drugs to interact with the target protein and precludes their respective functions. The other

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mechanisms by which C. albicans exhibits resistance to antifungal agents are active efflux of drugs out of the cell (Lupetti et al. 2002), which is mediated through upregulation of the efflux transport proteins: ABC transporter and major facilitator pumps (CDR and MDR) expressed on yeast cell membrane (Cannon et al. 2009). C. albicans biofilms are resistant to a variety of antifungal agents including the gold standards amphotericin B and fluconazole that are used for antifungal therapy (Chandra et al. 2001a; Ramage et al. 2001) which indicates that conventional antimicrobials have become unsuccessful to treat C. albicans-mediated infections (Ostrosky-Zeichner 2013). Although several groups have reported a few potential molecules like calcofluor white (Kingsbury et al. 2012), gymnemic acids (Vediyappan et al. 2013), phenazines (Morales et al. 2013), 4hydroxycordoin (Messier et al. 2011) and Solidago virgaurea water extract (Chevalier et al. 2012) for current treatment, the emergence of multidrug resistance in C. albicans still poses a significant challenge while treating C. albicans related infections. Hence to overcome these deficiencies, research on exploration of novel and effective compounds that: (1) reduce drug resistance genes, (2) prevent yeast–hyphal transition and (3) inhibit C. albicans biofilms is a worthy pursuit. In the present study, we have evaluated the potency of a few newly synthesized levofloxacin derivatives (Srinivasan et al. 2010) for their ability to suppress the above-mentioned virulence factors of C. albicans.

Materials and methods Preparation of standard yeast cell suspension C. albicans (ATCC: 90028) was maintained in Potato Dextrose (PD) (HiMedia) agar plates. Prior to all the assays, C. albicans was grown overnight in PD broth in Orbital shaker (Scigenics Biotech, Orbitek LE, India) at 75 rpm and 37 °C. At the late exponential growth phase, yeasts cells were harvested using a microfuge (Eppendorf, Germany) at 2,300 rcf for 15 min. Yeast cells were washed twice with phosphate buffered saline (PBS; pH 7.2, 0.1 M) and resuspended in PBS to reach an optical density (OD600) of 0.38 (107 cells ml−1) at 520 nm. From this, 100 μl of the suspension containing 107 cells ml−1 were used for all the assays (Seneviratne et al. 2009; Vandenbosch et al. 2010). Screening of levofloxacin derivatives for antibiofilm activity The yeast cell suspension of 100 μl (107 cells ml−1) was added to 1 ml of PD broth supplemented with the levofloxacin and their derivatives (6a, 6b, 6c, 6d, 7a, 7b and 7d) to yield a final concentration of the compounds ranging from 1 to 100 μg ml−1 in a 24-well polystyrene plate (Greiner Bio-

One). The plate was incubated for 48 h at 37 °C without agitation. After 48 h incubation, prior to the crystal violet assay, the wells containing the yeast cells suspension were analyzed spectrophotometrically (Shimadzu 2450, Japan) at 600 nm to ensure that there was no antibiotic activity. Decrease in the yeast cell density determines the antibiotic activity of the compound. In the crystal violet assay, the yeast cells and spent media were discarded and weakly adherent cells were removed by washing twice thoroughly with deionized water and the plates were dried before staining. C. albicans biofilm adhered to the polystyrene plates were stained with 400 μl of 0.4 % crystal violet solution (w/v) for 2–3 min. Subsequently, the dye was discarded and the wells were rinsed twice with deionized water. The wells were allowed to dry before solubilization of the crystal violet with 1 ml of absolute ethanol. The optical density was determined at 570 nm using the Multi label reader (Spectramax M3, USA). Qualitative analysis of C. albicans biofilm Scanning electron microscopy (SEM) analysis was performed for C. albicans biofilm in the presence and absence of levofloxacin derivatives at Biofilm Inhibitory Concentrations (BIC). The biofilm formed on the glass slides were fixed for 1 h in a solution containing 2.5 % glutaraldehyde. The glass pieces were washed in 0.1 M sodium acetate buffer (pH 7.3) and rinsed subsequently in MilliQ water. Later the glass slides were dehydrated in ethanol (70 % for 3 min, 95 % for 3 min and absolute ethanol for 5 min). The glass slides were dried, gold sputtered and examined with a Hitachi S-3000H (Japan) (Shafreen et al. 2011). Effects of levofloxacin derivatives on yeast–hyphal transition Morphological changes of C. albicansin the presence and absence of the levofloxacin derivatives was observed with acridine orange (AO) staining. C. albicans that were grown on the 24-well plates supplemented with the BIC of the compounds. The plates were incubated for 36 h at 37 °C with a slight agitation. Cells harvested after 36 h were washed twice and suspended in PBS. From this, 10 μl of the cell suspension was stained with 1 μl of the AO staining solution. The AO staining mixture was prepared with 100 mg ml−1 of AO in 1× PBS. The stained cells were visualized immediately under CLSM (Zeiss LSM710 meta, Germany). As mentioned above, standard cell suspension of C. albicans were grown on Spider medium containing 10 % fetal bovine serum (FBS), supplemented with and without the BIC of levofloxacin derivatives. The plates were incubated at 37 °C for 36 h. The morphology of C. albicans colony formed on the spider medium was visualized using the a gel documentation system (Bio-

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Rad Laboratories, XR+, USA) and the images were captured using Image Lab software (Tsang et al. 2012). Effects of levofloxacin derivatives on pre-formed C. albicans biofilm C. albicans biofilm was allowed to form on a glass slide (1× 1 cm) as mentioned above. The glass slides pre-formed with C. albicans biofilm were further incubated with BIC of the levofloxacin compounds for about 5 h. The treated slides were transferred to a 24-well plate and incubated for 45 min at 37 °C in 2 ml of PBS containing the fluorescein isothiocynate–concanavalin A (FITC–ConA; 50 μg ml−l) in 10 mmol−l hydroxyethylpiperazine ethanesulfonic acid (HEPES). Images of the stained slides were captured under CLSM and processed with Zeiss LSM Image Examiner (Version 4.2.0.121) (Carl Zeiss, Germany). For all the biofilm samples (control and treated) 20 Z-stack scans were performed and the CLSM images were exported as .LSM. Further, the CLSM images as biofilm stacks were analyzed using COMSTAT software (kindly gifted by Dr. Claus Sternberg, Technical University of Denmark). Three different features such as total biomass, maximum thickness and surface coverage area (SCA) were selected for further analysis (Zhao and Liu 2010). Quantitative PCR analysis for drug resistance genes The response of C. albicans drug resistance genes to the levofloxacin derivatives was quantified from the mRNA transcripts. For this, the assay mixture was prepared as mentioned above in the biofilm assay. The young biofilms after 24 h were harvested by centrifugation in RNAase free tubes (1.5 ml). The cell pellets were suspended in 1 ml of the TRIzol reagent (Sigma-Aldrich, Switzerland). Total RNA from both the control and treated samples were extracted using guanidine thiocyanate–phenol extraction method and RNA yield was quantified using BioSpecnano spectrophotometer (Shimadzu Corporation, Japan). The total RNA from the control and treated samples were reverse transcribed according to the manufacturer’s instruction, using cDNA reverse transcription kit (Applied Biosystems Inc., Foster, CA, USA). Real-Time PCR was carried out as per the manufacturer’s instructions using Power SYBR® Green PCR master mix (Applied Biosystems Inc., USA). The amplification data using the gene specific primers was composed by the ABI Sequence Detection 1.3 software (Applied Biosystems, USA). The data set was normalized with ACT1 (β-actin) gene from C. albicans (Table S1). Relative expression ratio (2−ΔΔCt) of the drug resistance genes (ERG11, MDR1, CDR1), in response to levofloxacin derivatives were quantified as fold

changes with the help of the following standard formula (Yuan et al. 2006). Normalization to endogenous control ΔC T sample ¼ C T Target gene−C T endogenous control ð1Þ

Normalization to calibrator sample ΔΔC T ¼ C T sample−C T endogenous control Ratio ¼ 2−ΔΔCt

ð2Þ ð3Þ

Quantification of ergosterol Alteration in ergosterol content with the presence and absence of levofloxacin derivatives were quantified using UVSpectrophotometer. As mentioned above, the yeast cell suspension (107 cells ml−1) was used to inoculate PDB supplemented with BIC of levofloxacin derivatives. The assay mixture was incubated at 37 °C for 48 h without shaking. The cells at OD600 =1.2 were harvested at 2,700 rpm for 5 min and washed once with distilled water, then dried and weighed. The cells were suspended with 3 ml of 25 % alcoholic potassium hydroxide and vortexed for 1 min. The tubes were incubated at 80 °C for 1 h and later were allowed to cool at room temperature. Subsequently, the total sterol was extracted with 1 ml of distilled water and 3 ml of n-heptane. The mixture was vortexed continuously for 10 min until the distinct layer of nheptane was observed. The clear layer of heptane was transferred to a clean borosilical tube. Further, the sterol extract (20 μl) was diluted up to 5-fold using 100 % ethanol and scanned spectrophotometrically between 200 and 300 nm with a UV spectrophotometer (Shimadzu 2450, Japan) (Shreaz et al. 2010). Statistical analysis Statistical comparisons among control and the treated samples were performed by using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). All the experiments were performed in triplicates and the comparative statistical difference was performed using paired sample t-test (when p was <0.05).

Results Determining the BIC of the levofloxacin derivatives Levofloxacin and their semi-synthetic derivatives (6a, 6b, 6c, 6d, 7a, 7b and 7d) were evaluated for their BIC (1 to 50 μg ml−1). Additionally, to check the efficiency of the

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compounds that produced significant reduction in C. albicans biofilm, the compounds were further diluted in milliQ water to 2- and 4-fold to perform the susceptibility test. Levofloxacin did not show any significant reduction of C. albicans biofilm, whereas the semi-synthetic derivatives tested against C. albicans showed a dose-dependent inhibitory effect on biofilm formation (Fig. 1a). Compounds 6a, 6b, 6c, 6d, 7b and 7d showed prominent effect by inhibiting the biofilms of C. albicans at low concentrations of 3–14 μg ml−1 (Table 1). The compound 7a did not show any antibiofilm activity even at a higher concentration of 50 μg ml−1. Furthermore, even at 100 μg ml−1 levofloxacin derivatives did not show any antifungal effect against C. albicans (Fig. 1b). Compounds 6a, 6c and 7d showing 50 % of antibiofilm activity at the least concentration of 3–7 μg ml−1 (BIC) were selected for further studies. Compound 7a without any inhibitory effect on C. albicans biofilm was used as the negative control for other assays.

Imaging of C. albicans biofilm SEM analysis was performed for the two best antibiofilm compounds (6a and 6c) observed from the crystal violet assay. The inhibitory effects of compounds 6a and 6c on C. albicans biofilms were comparatively monitored with the control biofilms formed in the absence of levofloxacin derivatives. C. albicans biofilms formed different morphological states with a combination of yeast cells and true hyphae (Fig. 2a). Fig. 1 a Effect of Levofloxacin derivatives at varying concentrations (1–14 μg ml−1) on C. albicans biofilm. b Effect of levofloxacin derivatives on growth of control and treated C. albicans. *Statistical significance (p<0.05) when compared with the untreated controls

Compounds 6a and 6c have completely reduced the attachment of the yeast cells to the glass slides (Fig. 2b, c). Effects of levofloxacin derivatives on C. albicans yeast to hyphal transition Morphology of the C. albicans in the presence and absence of the compounds was evaluated using fluorescent dye (AO). AO dye was used as a tool to stain the intact morphological features of C. albicans biofilm (Fig. 3). Levofloxacin derivatives (6a, 6c and 7d) completely prevented the hyphal growth which is an essential feature for biofilm formation (Fig. S1). C. albicans formed true hyphae in the absence of these compounds (Fig. 3a) and, on the other hand, Fig. 3b–d shows the intact yeast cells without the hyphal growth, when treated with levofloxacin derivatives. Effect of levofloxacin derivatives on C. albicans morphology was observed on spider medium. Figure 3e shows the presence of hyphal growth on the solid medium, whereas in the presence of BIC of 6a and 6c, the filamentous growth was effectively abrogated (Fig. 3f, g). From the above results, it is obvious that levofloxacin derivatives prevent the transition of yeast to hyphal, an essential characteristic of C. albicans virulence (Alem et al. 2006). Effects of levofloxacin derivatives on pre-formed C. albicans biofilm The architecture of pre-formed C. albicans biofilm was analyzed using CLSM micrographs. Figure 4a represents the

Appl Microbiol Biotechnol Table 1 Inhibitory effects of Levofloxacin derivatives on C. albicans biofilm at BIC Levofloxacin derivatives

BIC (μg ml−1)

% of Biofilm Inhibition

6a 6b 6c 6d

3 7 5 12

50.46 50.02 53.11 50.58

7b 7d

14 7

50.26 51.69

C. albicans biofilm that has attained maximum thickness (29.9 μm) and biomass (31.5 μm) by 48 h. When the preformed biofilms were incubated with levofloxacin derivatives for 5 h, compounds 6a, 6c and 7d dispersed the biofilm formed on the substratum which was evident from the decrease in thickness (6.2, 9.8 and 12.94 μm) and total biomass (10.0, 12.04 and 11.5 μm) (Fig. 4b–d). Contrary to expectation, compound 7a (10 μg ml−1), which did not show any antibiofilm activity in the preliminary experiments, showed significant dispersion of pre-formed C. albicans biofilms. The maximum thickness (11.2 μm) as well as biomass (17.6 μm) were significantly reduced upon treatment with 7a (Fig. 4e). The SCA in the treated samples was comparable to the control (Table 2).

Quantitative PCR analysis for drug resistance genes Treatment of young C. albicans biofilms with the 6a, 6c, 7d and 7a at BIC has resulted in a 2.8- to 1.4-fold decrease in ERG11 transcript level. Also, approximately a 1.5- to 1.0-fold decrease in expression of MDR and CDR1 were observed in the presence of levofloxacin derivatives. Thus from the comparative analysis it was observed that these compounds decreased the expression of two key membrane associated genes such as lanosterol 14-α demethylase (ERG11) involved in ergosterol biosynthesis pathway and membrane associated efflux transporter proteins (MDR and CDR1) (Fig. 5).

Quantification of ergosterol Sterols are essential features of the fungal cells in maintaining the integrity, fluidity and asymmetric structures of the membrane. Any change in sterol composition can alter the membrane biology and functions. Hence, the effects of levofloxacin derivatives on sterol composition were evaluated using UV spectrophotometer (Fig. 6a) and inhibition percentage was calculated. The samples treated with compounds 6a, 6c, 7d and 7a showed reduction of sterol content by about 19.50 %, 19.13 %, 15.78 % and 8.37 %, respectively (Fig. 6b), which implies the alternation in cellular process of C. albicans biofilms.

Discussion Formation of biofilms by C. albicans is observed as a protective environment for the cells to escape from antimicrobials agents (Donlan and Costerton 2002; Hawser and Douglas 1995). Antifungal agents (amphotericin B and azole groups) with clinical importance to treat fungal infections are less effective when used for treatment against C. albicans biofilm related infections. Therefore it is a dire need to search of novel anti candidal agents that prevent yeast–hypha morphogenesis, inhibit the penetration into the host cells and effectively disperse the biofilm. In the present study, levofloxacin derivatives reported from our laboratory with wide range of biological activities against different bacterial pathogens (Shafreen et al. 2011; Srinivasan et al. 2010) were evaluated against C. albicans biofilms. From the crystal violet assay, it was confirmed that levofloxacin derivatives significantly reduced C. albicans biofilms adhered to the polystyrene plates without inhibiting the growth, which is evident from the growth curve analysis (Fig. 1b). Levofloxacin did not show any considerable effect on C. albicans biofilm, whereas the derivatives show major effect on biofilm formation. Thus, from the structure activity relationship (SAR) it was observed that modification of the C-7 position of levofloxacin with the addition of N-aryl piperazine

Fig. 2 SEM micrographs showing the presence of hyphae in the thick biofilm of control (a) and the loosened biofilm upon treatment with the compound 6a (b) and 6c (c) at BIC. Scale bar=20 μm

Appl Microbiol Biotechnol Fig. 3 a CLSM of AO staining of C. albicans biofilm after 36 h. a Control with metabolically active hypha; b–d the intact yeast cells. b C. albicans spread on spider medium containing 10 % FBS in the presence and absence of levofloxacin derivatives: e control, f 6a and g 6c

Appl Microbiol Biotechnol Fig. 4 CLSM images for the control (a) and treated (6a [b], 6c [c], 7d [d] and 7a [e]) biofilm stained with fluorescein isothiocynate–concanavalin A (FITC–Con A)

(6a–6d) and piperidyl moiety (7a, 7b and 7d) is responsible for the broad range of antibiofilm activity (Fig. S2). From the analysis of SEM and CLSM, it was evident that the compounds at their BIC dramatically decreased biofilm formation. Also, from the SEM images, it was clear that compounds have disintegrated the biofilm architecture by loosening the yeast cells. The presence of the hyphae is characteristic of good biofilm architecture (Banerjee et al. 2012). Candida forms biofilms by (1) initial attachment of the yeast cell to the substratum, (2) followed with the germination of the hyphae, (3) development of the extracellular matrix and (4) finally

enters the maturation state (Nett and Andes 2006). Therefore, it is envisaged that compound 6a and 6c with excellent antibiofilm property inhibits the initial attachment of the yeast cells to the substratum and prevents the shifting of the yeast cells into later stages of biofilm development such as maturation and EPS production. In our earlier study (Shafreen et al. 2011), we have shown compound 6c inhibiting Streptococcus pyogenes biofilms up to 63 % at 2 μg of BIC. Mixed species biofilms on implant devices are less common than mono species biofilms but tend to be more serious and are an increasing problem. In a mixed species biofilm environment,

Appl Microbiol Biotechnol Table 2 COMSTAT analysis for biofilm formed by C. albicans in the presence and absence of the Levofloxacin derivatives C. albicans

Control

6a

6c

7d

7a

Biomass (μm3/μm2) Maximum thickness (μm) Surface coverage area (μm2/μm3)

31.5 29.9 0.056

1.5* 6.2* 0.051

2.04* 9.8* 0.052

1.0* 2.94* 0.050

27.6 27 0.041

bacterial cells produce EPS, which retards the penetration of the antifungal and antibacterial agents (fluconazole and vancomycin) (Adam et al. 2002; Pammi et al. 2013). Thus treating mixed species biofilms is even more challenging than mono species biofilms. As 6c showed antibiofilm activity against both bacteria and Candida species, we envisage that it can be an excellent candidate to treat mixed-species biofilms. The levofloxacin derivatives 6a and 6c are N-aryl piperazine derivatives which have 2-F-phenyl and 2-CNphenyl group, respectively. Thus, addition of these groups has enhanced the antibiofilm activity compared to the other levofloxacin derivatives. Furthermore, AO staining assay revealed that the compounds have significantly inhibited the hyphal formation which is clearly evident from the presence of only yeast cells (Fig. 3b–d). The levofloxacin derivatives 6a (3 μg ml−1) and 6c (5 μg ml−1) showed complete inhibition of the filamentous growth on the hyphal inducing medium supplemented with 10 % FBS. Hyphal formation in C. albicans is the integral component to maintain the architecture of Candida biofilms, which imposes serious medical challenges in immunocompromised patients (Morales et al. 2013). Based on this issue, in recent years several compounds were evaluated for their modulatory effect on C. albicans morphogenic states (Chevalier et al. 2012; Messier et al. 2011; Shareck and Belhumeur 2011; Toenjes et al. 2009; Tsang et al. 2012). C. albicans yeast-tohyphal conversion was impaired by 4-hydroxycordoin (50 μg ml−1), gymnemic acid (60 μg ml−1) and eugenol Fig. 5 Transcription analysis of ERG11 and drug resistance genes (CDR1 and MDR) in the presence and absence of levofloxacin derivatives. *p<0.05 statistically significant values when compared with the control and treated samples

(2,000 μg ml−1). Meanwhile, levofloxacin derivatives 6a, 6c and 7d used in the present study impaired the hyphal growth at very low concentrations (3–7 μg ml−1) when compared with the above compounds. Hyphal forms in C. albicans are an important feature for development of three-dimensional biofilm architecture. The hyphal forms also play a critical role in establishing pathogenicity by expressing hyphal cell wall proteins that penetrates deep into host tissues during infection (Vediyappan et al. 2013). As levofloxacin derivatives inhibited this critical step of yeast–hyphal transition (Fig. 3) we envisage that coating medical implants with levofloxacin derivatives may weaken the invasive capacity of C. albicans during device related infections where the implanted device is always in close contact with the host tissue. Continuous growth of pathogens on the substratum leads to the development of mature biofilm and enters the last stage of biofilm development known as biofilm dispersal. During biofilm dispersal bacterial cells detaches, enters a new location and colonize new sites. Hence, biofilm dispersal plays an important role in many pathogens with the transmission of the disease. Based on this issue, several compounds (Abee et al. 2011; Kaplan 2010; Nithya and Pandian 2010) have been investigated against different pathogens. To further explore the efficiency of levofloxacin derivatives as efficient antibiofilm agents, we attempted to evaluate the efficacy of the compounds to disperse mature biofilms. Application of levofloxacin derivatives on the pre-formed mature C. albicans biofilm resulted in pronounced biofilm dispersal. The CLSM images of control and treated biofilms were analyzed using the COMSTAT program. The statistical data revealed that addition of 6a, 6c, 7d and 7a compounds resulted in the decrease of thickness and biomass of C. albicans matured biofilms without any significant change in SCA of the control and treated mature biofilms. Therefore, from the present study it is envisaged that, in the presence of 6a, 6c, 7d and 7a the three-dimensional architecture of C. albicans mature biofilm was destroyed, and uniform monolayer of yeast cells attached to the substratum were exposed. Interestingly, compound 7a,

Appl Microbiol Biotechnol Fig. 6 C. albicans sterol profile a UV spectral profiles between 240 and 300 nm determined in presence and absence of the levofloxacin derivatives. b % inhibition of levofloxacin derivatives on total ergosterol content. *Statistically significant values (p<0.05)

which does not show any inhibitory effect on biofilm formation and morphogenic state of C. albicans, disrupted the preformed biofilm. SAR revealed the presence of an amide group attached to the piperidyl moiety of the levofloxacin derivative 7a. Therefore, we envisage that this amide group interacted with the exopolysaccharide and dispersed the pre-formed biofilms. The results of the present study assume greater significance in the context of the fact that well-known antifungal agents such as amphotericin B and miltefosine disperse mature biofilms of C. albicans at higher concentrations (2,000 and 1,600 μg ml−1, respectively; Vila et al. 2012) when compared to levofloxacin derivatives used in the present study. Thus, compounds 6a, 6c and 7a have a great potential to treat biofilm-associated infections. Furthermore, the effect of levofloxacin derivatives on the expression of drug resistance genes was investigated. During different stage of biofilm growth, young biofilms that show resistance to drugs are mainly due to the development of membrane associated efflux pumps of CDR and MDR (Mukherjee et al. 2003). Any mutation in ERG genes involved

in ergosterol biosynthesis leads to the alteration in the cellular process and overexpression of efflux pumps (Marichal et al. 1999; White et al. 1998). In the presence of levofloxacin derivatives 6a, 6c and 7d, C. albicans biofilms showed decreased expression of ERG11, CDR1 and MDR transcripts. Additionally, decrease in ergosterol profile was observed in the presence of levofloxacin derivatives. Inhibition of lanosterol 14-α demethylase diminishes the ergosterol content of the membrane and enhances the toxic sterol pathway intermediates. The alteration in the sterol content inhibits growth and leads to cell death (Akins 2005). Recently, cinnamaldehye and their synthetic derivatives have been reported with 58 % of ergosterol inhibition in C. albicans (Shreaz et al. 2010). Therefore, in the present study it is envisaged that the observed decrease in ergosterol content in the presence of levofloxacin derivatives could be due to the inhibition of C. albicans morphogenesis. In conclusion, the present study for the first time shows that levofloxacin derivatives are capable of preventing the biofilm formation and yeast–hyphal transition, the major virulence

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factors of C. albicans and we envisage that levofloxacin derivatives can form an alternative drug of choice to treat biofilm mediated implant device associated infections caused by C. albicans. Acknowledgments The authors acknowledge the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by DBT, GOI; Grant No. BT/BI/25/ 001/2006). The financial support provided in the form of SRF to RMBS by CSIR, HRDG (9/688(0012)/2011) is gratefully acknowledged.

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